JAC Advance Access published online on April 12, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn142
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Original research |
Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole
1 Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland 2 Swiss Tropical Institute, Socinstrasse 57, CH-4051 Basel, Switzerland
Received 27 November 2007; returned 30 January 2008; revised 27 February 2008; accepted 10 March 2008
* Corresponding author. Tel: +41-31-6312384; Fax: +41-31-6312477; E-mail: joachim.mueller{at}ipa.unibe.ch
Objectives: The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR.
Methods: In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle.
Results: Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 (‘antigenic shift’). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost.
Conclusions: These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.
Key Words: drug resistance , microarray analysis , differential gene expression , antigenic variation , encystation , excystation
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